The present invention relates to the use of human embryonic stem cells to create blood-related cells, and the use of those blood-related cells for various purposes.
Techniques for isolating stable cultures of human embryonic stem cells have recently been described by our laboratory. See U.S. Pat. No. 5,843,780 and J. Thomson et al., 282 Science 1145-1147 (1998). The disclosure of these publications and of all other publications referred to herein are incorporated by reference as if fully set forth below.
We have deposited two of our human embryonic stem cell lines with the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209 U.S.A. on Jul. 7, 1999 and Jul. 15, 1999 respectively (with accession numbers PTA-313 and PTA-353 respectively). These deposits are under the conditions of the Budapest Treaty. Taxonomic descriptions of these deposits are human embryonic stem cell lines H1 and H9 respectively. It has been proposed in these publications that such cell lines may be used for, among other things, providing a source of specified cell lines of various types for research, transplantation and other purposes.
Under the storage and culturing conditions described in these publications the cell lines are maintained long term without differentiation into specific cell types. When the cell lines are subsequently injected into immunodeficient mice, they form teratomas demonstrating differentiation into multiple tissue types.
When ES cells are used to produce desired cells, it is often preferable to optimize differentiation towards specific cell types. In the case of hematopoietic cells it is desirable that this result in hematopoietic cells that can be isolated and used to form multiple hematopoietic lineages. These cells may include, but not be limited to, hematopoietic stem cells.
Hematopoietic stem cell populations have been isolated directly from bone marrow. See C. Baum et al. 89 PNAS USA 2804-2808 (1992). However, this relies on a supply of bone marrow to obtain the cells.
There have also been some attempts to direct murine embryonic cell populations towards hematopoietic cells. See e.g. U.S. Pat. No. 5,914,268; G. Keller, 7 Current Opinion In Cell Biology, 862-869 (1995); and T. Nakano et al. 265 Science 1098-1101 (1994). See also M. Weiss, 11 Aplastic Anemia And Stem Cell Biology, 1185-1195 (1997); and S. Morrison et al., 11 Annu. Rev. Cell Dev. Biol., 35-71 (1995).
However, applying these teachings to primates has proven difficult. For example, in F. Li et al., 92 Blood 368a (1998) there was a discussion of techniques for differentiation of rhesus embryonic stem cell lines using a stromal cell line and exogenous cytokines. However, that group has more recently reported that their techniques had inadequate formation of colonies.
The treatment of various diseases by tissue transplantation has become routine. However, there can be waiting lists to obtain natural donated organs, cells, or tissue. Even when the natural donor material becomes available there is often a problem with rejection. Traditional approaches for suppressing an immune response of recipients have drawbacks. For example, immunosuppressive drugs are costly and often have side effects.
In WO 98/07841 there was discussed techniques of deriving embryonic stem cells that are MHC compatible with a selected donor (e.g. transplanting a donor nucleus into an enucleated oocyte, followed by derivation of the stem cells therefrom). The application suggested that the resulting cells could be used to obtain MHC compatible hematopoietic stem cells for use in medical treatments requiring bone marrow transplantation.
However, some diseases such as type 1 diabetes mellitus or multiple sclerosis involve an autoimmune response. For example, merely transplanting pancreatic islets (which are MHC compatible to the diseased individual) to replace destroyed pancreatic islets will not provide sufficient long term reduction in type 1 diabetes mellitus, as the immune system of the host will still attack the transplanted islets.
It can therefore be seen that a need exists for techniques for causing human embryonic stem cell cultures to differentiate to desired hematopoietic colonies. Further, it is desired to develop improved uses for hematopoietic cells.
In one aspect the present invention provides a method for obtaining human hematopoietic cells. One exposes a human embryonic stem cell culture to mammalian hematopoietic stromal cells so as to thereby create human hematopoietic cells. At least some of the human hematopoietic cells that are so created are CD34+ and/or are capable of forming hematopoietic cell colony forming units in methylcellulose culture.
CD34 is a standard marker for hematopoietic stem cells, as described in C. Baum et al. 89 PNAS USA 2804-2808 (1992) and S. Morrison et al., 11 Annu. Rev. Cell Dev. Biol., 35-71 (1995). The property of capability of forming a colony forming unit is indicative that the cells have the desired characteristics to form more differentiated hematopoietic lineages.
The stromal cells are preferably derived from bone marrow cells or embryonic yolk sac cells. Murine stromal cells may be used for this purpose. However, primate stromal and other mammalian stromal cells should be suitable as well.
In another aspect the invention provides a human hematopoietic cell which was derived from a human embryonic stem cell culture in vitro, and is capable of forming hematopoietic cell colony forming units in methylcellulose culture. As used in this patent, the term xe2x80x9cderivedxe2x80x9d is intended to mean obtained directly or indirectly (e.g. through one or more intermediates or passages).
In yet another aspect the invention provides a method of transplanting human cellular material into a human recipient host. One obtains human hematopoietic cells which have been derived in vitro from an embryonic stem cell culture. One then obtains a selected human cellular material other than hematopoietic cells, the selected non-hematopoietic material having major histocompatibility complex compatibility to the hematopoietic cells. One then transplants both the hematopoietic cells and selected human non-hematopoietic cellular material into the human host.
For example, one can obtain human hematopoietic cells which have been derived in vitro from an embryonic stem cell culture (e.g. using the techniques described below). One also obtains human pancreatic islets which have MHC compatibility to the hematopoietic cells. Both the hematopoietic cells and pancreatic islets are then transplanted into the human (preferably after the recipient""s own bone marrow has been inactivated).
The pancreatic islets can be obtained directly from a donor whose cells were used to create the embryonic stem cell culture. Alternatively, a single embryonic stem cell culture can be differentiated along two different paths. In one process the above technique can be used to create hematopoietic stem cells. These cells should develop into multiple hematopoietic lineages when transplanted into appropriate hosts. These lineages should include lymphocytes which would be tolerant of other cells derived from the same parental embryonic stem cells. In another process the stem cells would be directed towards pancreatic islets.
In another example one could supply oligodendrocytes to a human who has a multiple sclerosis condition. One obtains human hematopoietic cells which have been derived in vitro from an embryonic stem cell culture (e.g. using a technique described below). One also obtains human oligodendrocytes which have MHC compatibility to the bone marrow cells and transplants both the bone marrow cells and oligodendrocytes into the human.
The same human whose genetic material was used to create the embryonic stem cell can be a donor for the oligodendrocytes. Alternatively, the same embryonic stem cell culture can be differentiated along two separate paths to provide the two transplantable materials.
With respect to either disease (and potentially other autoimmune diseases) the immune and autoimmune rejection problems should be reduced by this technique. In this regard, the recipient""s original bone marrow can be totally or partially inactivated by radiation or chemical means before the transplantation. Thereafter, it is replaced at least in part by the transplanted hematopoietic cells. The elimination/reduction of the original bone marrow reduces the body""s ability to create an autoimmune response. The matching of the MHC of the replacement bone marrow and the second transplantable material insures that the second material won""t be rejected by the transplanted bone marrow.
Moreover, co-transplantation of hematopoietic cells and other tissue can be done to promote acceptance of the second tissue (e.g. heart muscle plus hematopoietic cells for treating heart disease; hepatocytes plus hematopoietic cells for treating liver disease). By creating hematopoietic chimeras improved acceptance of tissues with similarly matched MHC type can be obtained.
The present invention should be suitable to obtain a wide variety of hematopoietic cells of interest, such as erythroid cells, granulocyte cells, macrophages, lymphocyte precursors, monocytes, B cells, T cells, and the like. In this regard, colonies of differentiated ES cells develop into hematopoietic colonies when harvested, separated into single cells, and plated into appropriate cultures. These colonies demonstrate the development of colony-forming cells which proliferate into colony-forming units (including colony forming unit-erythroid (CFU-E), blast forming unit-eythroid (BFU-E), colony forming unit-macrophage (CFU-M), colony forming unit-granulocyte/macrophage (CFU-GM) and colony forming unit-high proliferative potential (CFU-HPP)). The identification of colony forming cells indicates the differentiation of embryonic stem cells into hematopoietic cells capable of expanding into defined hematopoietic lineages under defined conditions.
The objects of the present invention therefore include providing:
(a) methods of the above kind for obtaining hematopoietic cells;
(b) cells derived using those methods; and
(c) methods for using those derived cells for transplantation, transfusion and other purposes. These and still other objects and advantages of the present invention will be apparent from the description of the preferred embodiments that follows. However, the claims should be looked to in order to judge the full scope of the invention.